Iron is absolutely essential to sustain life and maintain growth of mammalian cells. Despite our fundamental knowledge of the utilization and storage of iron, however, relatively little is known about the translocation of the cation across biological membranes. The import of iron across cellular membranes must somehow be tightly controlled to guard against excessive assimilation while enabling the entry of adequate amounts of this essential nutrient. Project #3 focuses on a human K562 cell Fe transport protein, SFT, which we recently cloned by functional expression of non-transferrin-bound iron transport activity in Xenopus oocytes, and seeks to further our understanding of the function and mechanism of action of this novel factor. Interestingly, SFT not only mediates non-transferrin-bound iron uptake, but can also stimulate the acquisition of iron from transferrin as well. We will elucidate the mechanistic basis for SFT's effects on iron uptake mediated by the transferrin receptor. Possible interactions of SFT in the transferrin receptor-independent pathway also will be explored in Project #1. Our preliminary data further demonstrate that copper depletion blocks non-transferrin-bound iron uptake as well as transferrin-mediated transport stimulated by SFT. We seek to unravel the molecular basis for this effect. In this project, we focus our efforts on the requirement of copper for SFT-mediated uptake; it is anticipated that the synergy with Project #4 will reveal critical relationships between copper and various iron transport processes, including activities mediated by SFT. Finally, our preliminary results describe the high-affinity binding of extracellular iron by SFT. We will undertake a spectroscopic analysis of extra-membranous peptides of SFT and their interactions with iron. These studies will complement scanning mutagenesis approaches to identify functional domains of SFT.